1. Field
The disclosed subject matter generally relates to digital holographic microscopy.
2. Discussion of Related Art
Numerous biological samples, including live cells, are generally transparent under visible-light illumination and behave essentially as phase objects. Phase objects can only alter the phase component of optical waves, not their amplitude component. Hence, the phase structure of these objects are difficult to see with naked eyes or ordinary imaging techniques, which are only sensitive to the amplitude component of optical waves.
Phase imaging techniques can record phase structure of objects. For example, techniques such as Zernike's phase contrast (PC) method and Nomarski's differential interference contrast (DIC) method can visualize the phase component of optical waves by transforming the phase information into amplitude contrast. However the phase information provided by these techniques is only qualitative. As such, these techniques cannot be used to measure certain biological parameters of specimens, such as intracellular refractive indices, because measuring such biological parameters requires measuring phase structure of the biological specimen quantitatively.
These issues can be addressed using digital holographic microscopy (DHM). DHM is a non-destructive, full-field, label-free method of measuring the amplitude structure and the quantitative phase structure of microscopic specimens. In DHM, optical components are configured in such a way that a portion of coherent optical waves passes through a microscopic specimen, whereas the rest of the coherent optical waves propagates unhindered. The optical waves propagating through the specimens are called object waves, sometimes also called object beams, and the unhindered optical waves are called reference waves, sometimes also called reference beams. The structure of the microscopic specimen introduces amplitude attenuations and phase delays to the object waves with respect to the reference waves. In other words, the structure of the microscopic specimen is encoded in the object waves.
The information encoded in the object waves can be recovered by interfering the object waves with the reference waves and post processing. The reference waves and the object waves are interfered, and the interfered optical waves are recorded using an imaging device. The recorded interference pattern, also called an interferogram, is subsequently processed to reconstruct the amplitude and phase structure of the microscopic specimen.
In DHM, the reference waves and the object waves are interfered using one of the two general techniques: off-axis interferometry and on-axis interferometry. Off-axis interferometry interferes waves propagating along two different axes and records the resulting interferogram using a single camera exposure. The use of a single camera exposure renders this technique especially suited to imaging dynamic processes, such as cell growths, cell membrane fluctuations, cell swelling, neuronal activity, and cytoskeletal dynamics. Unfortunately, off-axis interferometry requires a spatial filtering during numerical reconstruction that separates the desired positive-order diffracted waves, and the unwanted zero- and negative-order diffracted waves. This spatial filtering process limits the spatial resolution of the reconstructed image.
In on-axis interferometry, on the other hand, the reference waves and the object waves propagate along the same axis, which allows for restoring the specimen information without using a resolution-limited spatial filter. However, on-axis interferometry requires recording at least three phase-shifted interferograms. Since a relatively long time is required for applying the phase shifts and acquiring the interferograms, this method of phase-shifting is not suitable for recording dynamic processes. In addition, on-axis phase-shifting interferometry is also prone to phase-shift calibration errors, and system fluctuations that may occur while capturing multiple interferograms.
Some of these issues are partly addressed in Zhang et al., entitled “Reconstruction of in-line digital holograms from two intensity measurements.” Zhang shows an optical system that records two interferograms of an amplitude object (i.e., an object that attenuates only the amplitude of illuminated optical waves) and processes the two interferograms to recover an image of the amplitude object.
However, Zhang's optical system is tailored to reconstructing amplitude objects only and is incapable of reconstructing phase objects. Zhang et al. assume that the intensity of the object waves is significantly weaker than that of the reference waves. While this assumption might hold for objects with low transmission coefficients such as amplitude objects, this assumption does not hold for objects with high transmission coefficients such as phase objects. Therefore, Zhang's optical system is incapable of imaging object with both amplitude and phase structures.
FIG. 4 experimentally shows that Zhang's system fails to recover the phase structure of phase objects, and FIG. 6 experimentally shows that Zhang's system also does a poor job in recovering the amplitude structure of amplitude objects due to the impractical weak-object wave assumption. Therefore, Zhang's optical system cannot accurately recover either the amplitude structure or the phase structure of specimens. Thus, it is desirable to develop an in-line DHM technique that is capable of capturing amplitude and phase structures of microscopic objects while maintaining a short exposure time, a high spatial resolution, and a high contrast.